Characterization of the SAMHD1-mediated restriction to lentiviral infection counteracted by Vpx and analyzing its role in innate immunity

在慢病毒与宿主免疫系统的战斗中，非结构蛋白已经成为关键角色。HIV-2/SIVmac/SIVsm谱系的病毒除了其近亲Vpr外，还编码非结构蛋白Vpx。Vpx，而不是Vpr，促进SIV和HIV-1感染的单核细胞源性巨噬细胞（MDM）和树突状细胞（DC），其中它被认为是中和一个未知的限制因子。最近，SAMHD1已被确定为推测的限制因子，并与Vpx表型相关。髓系限制的机制尚不清楚，但磷酸水解酶结构域的重要性表明，其假定的核酸酶活性可能是关键。由于SAMHD1在不同的细胞系中表达，它很可能不能决定阻断的髓细胞特异性。本项目将使用基因转移和蛋白质组学方法来鉴定SAMHD1潜在的髓细胞特异性辅助因子。我们还将描述SAMHD1阻滞的机制，并分析Vpx与SAMHD1的结合。据报道，Vpx克服这一阻断会引发DC患者对HIV-1的先天免疫反应。我们将确定这种效应是DC独有的，还是也存在于MDM中，MDM是HIV-1在体内的主要靶细胞。了解这种免疫反应和Vpx在感染中的作用可能会回答为什么HIV-1只编码Vpr而不编码Vpx的问题。Vpx-SAMHD1的相互作用也将为Vpr提供线索，Vpr很可能通过类似的途径发挥作用。对Vpx和Vpr在先天免疫中的作用的了解可能会揭示感染中的一个脆弱步骤，这可能是抗病毒药物开发的目标。

Non-structural proteins have emerged as key players for lentiviruses in their battle with the host immune system. Viruses of the HIV-2/SIVmac/SIVsm lineage encode, in addition to its close relative Vpr, the non-structural protein Vpx. Vpx, but not Vpr facilitates the SIV and HIV-1 infection of monocyte-derived macrophages (MDM) and dendritic cells (DC), where it has been proposed to neutralize an unknown restriction factor. Recently, SAMHD1 has been identified as the putative restriction factor and linked to the Vpx phenotype. The mechanism of the myeloid restriction is not clear but the importance of the phosphohydrolase domain suggests that its putative nuclease activity might be key. Since SAMHD1 is expressed in divergent cell lines, it is most likely not determining the myeloid cell specificity of the block. This project will use gene transfer and proteomics approaches to identify potential myeloid cell specific cofactors of SAMHD1. We will also characterize the mechanism of the SAMHD1 block and analyze the binding of Vpx to SAMHD1. It has been reported that overcoming this block by Vpx triggers an innate immune response to HIV-1 in DC. We will determine whether this effect is unique to DC or whether it is also present in MDM, which are main target cells of HIV-1 in vivo. Understanding this immune response and the role of Vpx in infection might answer the question why HIV-1 encodes only Vpr and not Vpx. The Vpx-SAMHD1 interaction will also provide clues to Vpr, which most likely functions through a similar pathway. The insights on the role of Vpx and Vpr in innate immunity may unearth a vulnerable step in infection that could be targeted by antiviral drug development.